Regulation of Kir2.1 channels by the Rho-GTPase, Rac1.
Mutations in Kir2.1 inwardly rectifying potassium channels are associated with Andersen syndrome, a disease characterized by potentially fatal cardiac arrhythmias. While several Andersen-associated mutations affect membrane expression, the cytoplasmic signals that regulate Kir2.1 trafficking are poorly understood. Here, we investigated whether the Rho-family of small GTPases regulates trafficking of Kir2.1 channels expressed in HEK-293 cells. Treatment with Clostridium difficile toxin B, an inhibitor of Rho-family GTPases, or co-expression of the dominant-negative mutant of Rac1 (Rac1(DN)) increased Kir2.1 channels approximately 2-fold. However, the dominant-negative forms of other Rho-family GTPases, RhoA or Cdc42, did not alter Kir2.1 currents, suggesting a selective effect of Rac1 on Kir2.1 channels. Single-channel properties (gamma, tau(o), tau(c)) and total protein levels of Kir2.1 were unchanged with co-expression of Rac1(DN); however, studies using TIRF microscopy and CFP-tagged Kir2.1 revealed increased channel surface expression. Immunohistochemical detection of extracellularly tagged HA-Kir2.1 channels showed that Rac1(DN) reduced channel internalization when co-expressed. Finally, the dominant-negative mutant of dynamin, which interferes with endocytosis, occluded the Rac1(DN)-induced potentiation of Kir2.1 currents. These data suggest that inhibition of Rac1 increases Kir2.1 surface expression by interfering with endocytosis, likely via a dynamin-dependent pathway. Surprisingly, Rac1(DN) did not alter Kir2.2 current density or internalization, suggesting subunit specific modulation of Kir2.1 channels. Consistent with this, construction of Kir2.1/2.2 chimeras implicated the C-terminal domain of Kir2.1 in mediating the potentiating effect of Rac1(DN). This novel pathway for regulating surface expression of cardiac Kir2.1 channels could have implications for normal and diseased cardiac states.
Boyer
Stephanie B
SB
Peptide Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA. sboyer@ucsd.edu
Slesinger
Paul A
PA
Jones
S V Penelope
SV
D002460:Cell Line; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis; D005799:Genes, Dominant; D006801:Humans; D015640:Ion Channel Gating; D008856:Microscopy, Fluorescence; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015854:Up-Regulation; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
C489461:Kir2.1 channel; C489463:Kir2.2 channel; D024661:Potassium Channels, Inwardly Rectifying; D020830:rac1 GTP-Binding Protein; D034281:Dynamins